Purpose:
We investigated the accumulation of amyloid β (Aβ1–40, Aβ1–42, Aβ1–43) in the lens epithelium of patients with opacification of five different types (cortical cataract [COR]; nuclear cataract [NUC]; posterior subcapsular cataract [PSC]; retrodots [RD]; and water clefts [WC]).

Methods:
Samples were collected from Japanese patients taken during cataract surgery; Aβ levels and mRNA expression were determined by ELISA and a real-time RT-PCR method, respectively.

Results:
Levels of Aβ1–40 and Aβ1–42 in the lens epithelium of patients with COR, NUC, PSC, RD, and WC showed no significant differences in comparison with transparent lens epithelium. Levels of Aβ1–43 in the lens epithelium of patients with PSC and WC were not detected, and NUC and RD were slightly elevated. In contrast to the results in these cataract types, high Aβ1–43 levels were observed in the lens epithelium of patients with COR, and a close relationship was observed between Aβ1–43 levels and the degree of lens opacification (R = 0.8229, n = 6). The levels of Aβ1–43 were also higher in the lens epithelium of patients with mixed-cataract showing cortical opacification, and the Aβ1–43 levels in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in that of mixed-cataract patients without cortical opacification. In addition, the level of an amyloid precursor protein mRNA in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in transparent lens and mixed-cataract patients without cortical opacification.

Conclusions:
We found high levels of Aβ1–43 accumulation in the lens epithelium of Japanese patients with cortical opacification.

Age-related cataracts are the most common cause of vision loss in the elderly, and the principal cause of blindness in the world. Age-related cataracts are classified by the location of the opacity within the lens: cortical cataracts (COR); nuclear cataracts (NUC); and posterior subcapsular cataracts (PSC). In addition, retrodots (RD) and water clefts (WC) have been found to be associated with visual impairment. Cataracts represent a complex disease with numerous genetic and environmental contributing factors, including smoking, ultraviolet irradiation, diabetes, the cumulative effect of X-rays, nutrition, vitamin C deficiency, hypertension, and alterations in both endocrine and enzymatic equilibria.1–8 On the other hand, it has recently been reported that the accumulation of amyloid β (Aβ) peptides in the human lens may be related to the onset of lens opacification,9–11 but this involvement in terms of etiology and Aβ levels is not fully understood.12

The enzymes α-secretase (a disintegrin and metalloproteinase, ADAM10)13–15; β-secretase (β site APP cleaving enzyme, called BACE1)16; and γ-secretase (a presenilin complex, PS1 and PS2)17 are known to be related to Aβ production by cleaving an amyloid precursor protein (APP). The peptides produced by PS and ADAM10 are short peptides that are nontoxic. On the other hand, Aβ produced by the sequential proteolytic processing of APP by BACE1 and PS, are peptides that show toxicity. Peptides Aβ are classified as Aβ1–40, Aβ1–42, and Aβ1–43 peptides,18–22 and their aggregation, amounts, and toxicity differ. It has been reported that the amount of Aβ1–40 is higher than those of Aβ1–42 or Aβ1–43; however, the Aβ1–42 and Aβ1–43 peptides aggregate more strongly than Aβ1–40.22–24 Although the toxicity of Aβ1–43 is higher than that of Aβ1–40 or Aβ1–42, Aβ1–43 accumulation levels are lower than those of Aβ1–42.22 Under normal conditions, the Aβ peptide is cleaved and degraded by peptidases such as neprilysin (NEP),25 and endothelin converting enzyme (ECE).26 In the present study, we investigated the accumulation of Aβ1–40, Aβ1–42, and Aβ1–43 in the lens epithelium of patients with 5 different types of cataracts (COR, NUC, PSC, RD, and WC). In addition, we demonstrate a relationship between Aβ accumulation and cortical opacification in human lens epithelium with mixed cataracts.

Methods

Collection of Human Lens Samples

Table 1 shows the number of cataract patients in each cataract group. These samples of normal donor lens epithelium of Japanese patients (normal) obtained by cataract surgery combined with vitrectomy for epiretinal membrane or macular hole were used as noncataractous controls (clear, age 62.8 ± 3.6 years, n = 19 [male: 9, female: 10]). And the opaque lens epithelium of Japanese patients (age 72.7 ± 1.1 years, opacity score 3.91 ± 0.21, n = 78 [male 44, female 34]) were collected from patients undergoing cataract surgery. These normal and opaque samples were provided from nondiabetes mellitus, non-Alzheimer's patients at Kanazawa Medical University (Ishikawa, Japan), and immediately stored in liquid nitrogen or RNA stabilization solution (RNAlater; Qiagen, Tokyo, Japan) until use. The enucleated lens epithelium samples were classified according to cataract type: COR; NUC; PSC; RD; WC; cortical opacification with or without nuclear opacification; posterior subcapsular opacification; RD and WC (mixed-cataracts with cortical opacification); and opacification with or without nuclear opacification, posterior subcapsular opacification, RD and WC (mixed-cataracts without cortical opacification). Patients with cataracts were screened for visual acuity in the clinic prior to surgery; cataract types were determined according to the WHO classification and Kanazawa Medical University classification criteria.27 All procedures were performed in accordance with the Kanazawa Medical University Research Ethics Committee (No. 96), and Kindai University School of Pharmacy Committee for Research Ethics (No. 13-046).

Samples of lens epithelium were collected from patients undergoing cataract surgery at Kanazawa Medical University (Ishikawa, Japan), and immediately stored in RNA stabilization solution (Qiagen) until use. Total RNA was extracted from the lens epithelium using an RNA extraction kit (RNeasy Micro; Qiagen), and the RT-PCR reaction was performed using an RNA PCR kit (AMV version 3.0; TaKaRa Bio, Inc., Shiga, Japan) and a master mix (LightCycler FastStart DNA Master SYBR Green I; Roche Molecular Systems, Inc., Indianapolis, IN, USA) according to our previous report.28Table 2 shows the specific primers (10 pmol) used in this study. The conditions for PCR were as follows: hot start, 95°C for 10 minutes; denaturing, 60 cycles of 95°C for 10 seconds; annealing, 63°C for 10 seconds; extension, 72°C for 5 seconds. The differences in the threshold cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other groups (APP, ADAM10, BACE1, PS1, PS2, NEP and ECE-1) were used to calculate the levels of mRNA.

All data are expressed as the mean ± SE of the mean. Statistical significance was evaluated using the Student's t-test, Aspin-Welch's t-test, or Dunnett's multiple comparisons.

Results

Aβ Accumulation in Lens Epithelium of Five Different Cataract Type

Before we investigated the relationship between Aβ accumulation and lens opacification, we confirmed that Aβ levels in lens epithelium do not change with age (Fig. 1). In addition, the Aβ levels in lens epithelium of patient with cataract were also similar between the male and female (Table 3). Figure 2 shows changes in total Aβ, Aβ1–40, Aβ1–42 and Aβ1–43 levels in lens epithelium according to cataract type (COR, NUC, PSC, RD, and WC). Total Aβ, Aβ1–40, and Aβ1–42 levels showed no significant differences between normal donor transparent lenses and lenses from patients with COR, NUC, PSC, RD, or WC type cataracts. In contrast to total Aβ, Aβ1–40, and Aβ1–42 levels, Aβ1–43 was not detected in lenses from normal donors or in patients with PSC or WC cataracts. Although Aβ1–43 was detectable in lenses from patients with NUC and RD cataracts, the levels were at the lower limit of quantitation for the ELISA kit used. On the other hand, high Aβ1–43 levels were detected in patients with COR cataracts, and the levels were 18.5- and 13.9-fold higher as compared with patients with NUC and WC cataracts, respectively. Figure 3 shows changes in the Aβ1–40/total Aβ, Aβ1–42/total Aβ, and Aβ1–43/total Aβ ratios in the lens epithelium of patients with different cataract types (COR, NUC, PSC, RD, and WC). Although the Aβ/total Aβ ratios for patients with NUC, PSC, RD, or WC cataracts were similar to those for normal donor lens, the Aβ1–43/total Aβ ratio in patients with COR cataracts was clearly higher than that for normal donors. In addition, opacification levels in patients with COR cataracts increased with the increase in Aβ1–43 levels (Fig. 4).

Figures 5 and 6 show the changes in total Aβ, Aβ1–40, Aβ1–42, Aβ1–43 levels (Fig. 5) and Aβ1–40/total Aβ, Aβ1–42/total Aβ, Aβ1–43/total Aβ ratios (Fig. 6) in lens epithelium from mixed-cataract patients with or without cortical opacification. Total Aβ, Aβ1–40 and Aβ1–42 levels as well as Aβ1–40/total Aβ and Aβ1–42/total Aβ in lenses from mixed-cataract patients with or without cortical opacification did not differ significantly from those in normal donor lenses. As to Aβ1–43, no Aβ1–43 expression was detected in lens epithelium from normal donors, and only slight Aβ1–43 accumulation in samples from mixed-cataract patients without cortical opacification. On the other hand, high Aβ1–43 levels were observed in mixed-cataract patients with cortical opacification: the Aβ1–43/total Aβ ratio and Aβ1–43 level in mixed-cataract patients with cortical opacification were 13.6- and 26.1-fold higher than in mixed-cataract patients without cortical opacification, respectively. Figure 7 shows the expression levels of APP, ADAM10, BACE1, PS, NEP, and ECE-1 mRNA in lens epithelium from mixed-cataract patients with or without cortical opacification. Although there were no significant differences in the expression levels of BACE1, PS, ADAM, NEP, and ECE1 mRNA among normal donor and mixed-cataract patients with or without cortical opacification, the APP mRNA expression level in mixed-cataract patients with cortical opacification was significantly higher than in both normal donors and mixed-cataract patients without cortical opacification.

In a previous study, Goldstein et al.9 showed that Aβ is present in the cytosol of lens fiber cells of people with Alzheimer's disease, and that Aβ in the lens promotes regionally specific lens protein aggregation and supranuclear cataracts. Moncaster et al.10 also reported enhanced Aβ expression in a lens from a Down syndrome patient that resulted in amyloidogenic interactions with αB-crystallin within the cytoplasm of supranuclear lens fiber cells leading to increased lens opacification, light scattering, and protein aggregation. On the other hand, Michael et al.12 reported not being able to detect Aβ in lens cortex of an Alzheimer disease patient. From these reports, the question of whether or not Aβ accumulates in cataractous human lens is currently controversial. It is known that the epithelial cells on the anterior surface of the lens move toward the lens nuclear portions during growth, and our previous report showed that that part of the lens epithelium is related to Aβ production.28 Therefore, we investigated the accumulation of Aβ1–40, Aβ1–42, and Aβ1–43 levels in lens epithelium from Japanese patients taken during cataract surgery in order to clarify the relationship between Aβ accumulation and lens opacification. The methods, that would yield exact and reproducible results, were needed because Aβ levels (especially Aβ1–43) in the lens are remarkably low. Previous reports by the Goldstein et al.9 and other laboratories (Frederikse et al.29) showed that the mass spectrometry was useful to identify the Aβ. On the other hand, a highly sensitive ELISA method was also developed, and further development for Aβ study is expected. In this study, the Aβ1–40, Aβ1–42, and Aβ1–43 levels in cataractous lens epithelium were measured using the highly sensitive ELISA method.

First, we investigated Aβ1–40, Aβ1–42, and Aβ1–43 accumulation in lens epithelium with different cataract types (COR, NUC, PSC, RD, or WC). In normal donor transparent lens, Aβ1–40 and Aβ1–42 accumulation was detected, while Aβ1–43 accumulation was not observed (Fig. 2). The accumulation of Aβ1–40 and Aβ1–42 was also observed in patients with COR, NUC, PSC, RD, or WC type cataracts at levels similar to those in normal donor lens. Accumulation of Aβ1–43 was not detectable in patients with PSC and WC type cataracts, and very low Aβ1–43 levels, at the lower limit of quantitation by the ELISA kit used, were detected in patients with NUC and RD type cataracts. In contrast to the findings in these cataract types, high Aβ1–43 levels in patients with COR type cataracts were observed in comparison with normal donors (Fig. 2); and the Aβ1–43/total Aβ ratio was also higher than in normal donors or patients with COR, NUC, PSC, RD, or WC type cataracts (Fig. 3). In addition, a close relationship was observed between the Aβ1–43 level and the degree of lens opacification in patients with COR type cataracts (Fig. 4). These results show that the accumulation of Aβ1–43 in lens epithelium leads to cortical opacification in Japanese patients, and patients with a predisposition to accumulate the Aβ1–43 in lens may experience cortical opacification. Moreover, there appears to be large variability in the data between age 60 and 75 years when a correlation between cortical opacity and Aβ might be most meaningful (Figs. 1, 4). The mechanism of Aβ1–43 expression will be examined in a future study.

Next, we examined lens epithelium from patients whose cataracts involved opacification in the cortex and a different area (mixed-cataract patients with cortical opacification) and from patients whose cataracts involved opacification in multiple areas other than the cortex (mixed-cataract patients without cortical opacification). The levels of Aβ1–43 expression and accumulation observed in lens epithelium from mixed-cataract patients with cortical opacification were both significantly higher than those in the lens epithelium from mixed-cataract patients without cortical opacification (Figs. 5, 6). Moreover, we measured the gene expression levels for proteins related to Aβ production in lens epithelium of mixed-cataract patients with or without cortical opacification, and found that the expression of APP mRNA was significantly higher in mixed-cataract patients with cortical opacification than in normal donors or mixed-cataract patients without cortical opacification (Fig. 7). It is known that reactive oxygen species (ROS) such as hydrogen peroxide are related to the onset of cortical opacification,30–35 and there have been several reports that hydrogen peroxide leads to increases in the production and accumulation of Aβ peptides in neuronal cells such as retina and brain, and that Aβ enhances oxidative stress via ROS.30–35 We have also reported that the stimulation of ROS hydrogen peroxide augmented gene expression of the proteins related to Aβ production, resulting in the production of three types of Aβ peptides (Aβ1–40, Aβ1–42, and Aβ1–43) in the human lens epithelial SRA01/04 cells.36 In addition, we used lens epithelium located on the outer surface of the lens in this study. Taken together, we hypothesize that ROS via ultraviolet irradiation causes an increase in APP mRNA expression in the lens epithelium of mixed-cataract patients with cortical opacification, and that this enhanced APP mRNA expression may be related to the accumulation of Aβ1–43 peptides in patients with cortical opacification. On the other hand, enhanced APP mRNA expression did not induce increases in Aβ1–40 or Aβ1–42 levels in the lens epithelium of patients with COR type cataracts. The accumulation of both Aβ1–40 and Aβ1–42 was detected in lenses from normal donors, while Aβ1–43 accumulation was not observed (Fig. 2). Therefore, the measurement of Aβ1–43 may be a sensitive way to detect changes in comparison with measuring Aβ1–40 and Aβ1–42 since the Aβ1–43 abundance ratio in the lens is lower than those of Aβ1–40 or Aβ1–42. On the other hand, Aβ1–43 expression was not detected in 6 out of 25 samples from mixed-cataract patients with cortical opacification in our study, and the opacity score in the cortical area of mixed-cataract samples in which Aβ1–43 was detected (1.59 ± 0.17) was lower than that of mixed-cataract samples in which Aβ1–43 was not detected (2.25 ± 0.25). These results show that the accumulation of Aβ1–43 is one factor involved in the onset of cortical opacification; however, Aβ1–43 is not a factor in all cases of cortical opacification.

In conclusion, we have found high levels of Aβ1–43 accumulation in lens epithelium from Japanese patients with cortical opacification. On the other hand, it is unclear just how Aβ1–43 is produced and what its significance might be for the lens and for cataract. Recently, it has been suggested that the mixtures of Aβ1–42 and Aβ1–43 aggregate more slowly than Aβ1–42 alone. Both in this Aβ1–42/Aβ1–43 coaggregation reaction and in cross-seeding by Aβ1–42 fibrils, the structure of the Aβ1–43 in the product fibrils is influenced by the presence of Aβ1–42.37 In addition, copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy human lens, and a variety of experimental and epidemiologic studies implicate metals as potential etiologic agents for cataract patients.38 The increased intracellular copper or zinc has been linked to a reduction in secreted levels of the Aβ.39 Moreover, the zinc has clear antioxidant properties and prevented oxidative stress by scavenging free radicals generated by ionizing radiation in rat lenses.40 Taken together, further studies are needed to elucidate the precise mechanism for the accumulation and toxicity of Aβ1–43 in the lenses of patients with cortical opacification. In addition, it is important to clarify the precise relationships of Aβ and copper or zinc in the lens epithelium. Therefore, we are now investigating the effect of soluble or insoluble Aβ1–43 accumulation on lens opacification using such animal models of cortical opacification, diabetic cataract models and human lens epithelial cells.